Adenosine Derived From ADP Can Contribute to Inhibition of Platelet Aggregation in the Presence of a P2Y ₁₂ Antagonist

Abstract

Objective-To investigate whether adenosine diphosphate (ADP)-derived adenosine might inhibit platelet aggregation, especially in the presence of a P2Y 12 antagonist, where the effects of ADP at the P2Y 12 receptor would be prevented. Methods and Results-Platelet aggregation was measured in response to thrombin receptor activator peptide by platelet counting in platelet-rich plasma (PRP) and whole blood in the presence of ADP and the P2Y 12 antagonists cangrelor, prasugrel active metabolite, and ticagrelor. In the presence of a P2Y 12 antagonist, preincubation of PRP with ADP inhibited aggregation; this effect was abolished by adenosine deaminase. No inhibition of aggregation occurred in whole blood except when dipyridamole was added to inhibit adenosine uptake into erythrocytes. The effects of ADP in PRP and whole blood were replicated using adenosine and were directly related to changes in cAMP (assessed by vasodilator-stimulated phosphoprotein phosphorylation). All results were the same irrespective of the P2Y 12 antagonist used. Conclusion-ADP inhibits platelet aggregation in the presence of a P2Y 12 antagonist through conversion to adenosine.Inhibition occurs in PRP but not in whole blood except when adenosine uptake is inhibited. None of the P2Y 12 antagonists studied replicated the effects of dipyridamole in the experiments that were performed. Key Words: antiplatelet drugs Ⅲ arterial thrombosis Ⅲ blood cells Ⅲ cell physiology Ⅲ g proteins Ⅲ hemostasis Ⅲ platelet receptor blockers Ⅲ platelets Ⅲ thrombosis Ⅲ P2Y 12 antagonists Ⅲ adenosine A DP is a well-known platelet agonist that induces platelet aggregation. It is secreted from platelets themselves when they undergo a release reaction and potentiates the aggregatory effects of other platelet agonists, such as thrombin and collagen. [1][2][3] The proaggregatory effects of ADP on platelets are via interaction with P2Y 1 and P2Y 12 receptors, 1-4 and one of the main effects of ADP at the P2Y 12 receptor is the inhibition of adenylate cyclase, leading to a reduction in cAMP levels and subsequent promotion of the aggregation response. Antagonists that act at P2Y 12 receptors markedly inhibit platelet aggregation, specifically by blocking the effects of ADP at this receptor, 5 thereby preventing inhibition of adenylate cyclase. Such antagonists include cangrelor, ticagrelor, and the thienopyridines clopidogrel and prasugrel, all of which are in use or in development as antithrombotic agents. 6 -8 Although ADP is conventionally regarded as a platelet agonist, the molecule can be broken down first to AMP and then to adenosine in blood and plasma. 9 -11 Adenosine, of course, is an inhibitor of platelet aggregation acting via A 2A and A 2B receptors on platelets to activate adenylate cyclase and increase cAMP, 12-14 and both ADP removal and adenosine production might be expected to modulate platelet function. [15][16][17][18][19][20] Such modulation may be particularly evident in the presence of a P2Y 12 antagonist that blocks the ability of the ADP to inhibit ad...

Ticagrelor inhibits human platelet aggregation via adenosine in addition to P2Y12 antagonism

“…Our results are in disagreement with a previous report by Iy u et al [25], in which dipyridamole, but not ticagrelor, potentiated the inhibitory effect of exogenous adenosine on platelet aggregation. There are many methodological differences including the method to measure platelet aggregation in whole blood (Multiplate vs. single platelet counting), the type of platelet agonist (collagen vs. thrombin receptor activating peptide) and the observa- figure).…”

P2Y12and EP3 antagonists promote the inhibitory effects of natural modulators of platelet aggregation that act via cAMP

“…However, in the presence of dipyridamole, an inhibitor of ENT [34][35][36][37], both adenosine and ADP brought about inhibition of platelet function as in PRP. That adenosine is indeed the mediator of inhibition of platelet function was confirmed using ADA which prevented the inhibitory effects of adenosine and ADP [29]. In the experiments described here, we looked at the potency of adenosine acting as an inhibitor of platelet aggregation in whole blood containing dipyridamole, and showed that in the presence of a P2Y 12 antagonist that potency was increased in the same way as for the prostaglandins that we investigated.…”

“…In a previous publication [29], we have already shown that P2Y 12 antagonists enhance the inhibitory effects of adenosine. Indeed, concentration-dependent inhibition of platelet function was also seen with ADP in the presence of a P2Y 12 antagonist consequent to conversion of the ADP to adenosine.…”

“…Samples were then frozen and stored at À20 C prior to assay. The amounts of the phosphorylated form of VASP (VASP-P) present in the lysates were measured using a flow cytometric bead assay which has been described previously [25,[27][28][29]. Briefly, platelet lysates (25 ml) were incubated with APC-fluorescent beads (Becton Dickinson) (25 ml) coated with a monoclonal antibody to VASP (mAb IE273 anti-VASP conjugated using the Functional Bead Conjugation Buffer Set).…”

The concordance and correlation of measurements by multiple electrode and light transmittance aggregometries based on the pre-defined cutoffs of high and low on-treatment platelet reactivity

“…Additionally, PA in MEA takes place on surface similar with ruptured plaques or vascular injury [7]. However, if MEA used whole blood drawn from patients on ticagrelor or phosphodiesterase inhibitor, the plasma adenosine is rapidly taken up by erythrocytes in whole blood, and this ex vivo measurement can underestimate the actual in vivo antiplatelet effect of these agents [26]. Moreover, the results of MEA-based studies predicting the risk of clinical events were contradictory: some studies provided good predictive values [11][12][13], but some did not [14,27].…”